Penicillium discolor, a new species from cheese, nuts and vegetables

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1 Antonie van Leeuwenhoek 72: , c 1997 Kluwer Academic Publishers. Printed in the Netherlands. Penicillium discolor, a new species from cheese, nuts and vegetables Jens C. Frisvad 1, Robert A. Samson 2, Birgitte R. Rassing 1, Marjolein I. van der Horst 2, F.T.J. van Rijn 3 &J.Stark 3 1 Department of Biotechnology, Building 221, Technical University of Denmark, DK-2800 Lyngby, Denmark; 2 Centraalbureau voor Schimmelcultures, P.O. Box 273, NL-3740 AG Baarn, The Netherlands, 3 Gist-brocades, P.O. Box 1, 2600 MA Delft, The Netherlands; Author for correspondence (Fax Accepted 2 December 1996 Key words: Penicillium discolor sp. nov., cheese, nuts, chaetoglobosin Abstract The new species Penicillium discolor, frequently isolated from nuts, vegetables and cheese is described. It is characterised by rough, dark green conidia, synnemateous growth on malt agar and the production of the secondary metabolites chaetoglobosins A, B and C, palitantin, cyclopenin, cyclopenol, cyclopeptin, dehydrocyclopeptin, viridicatin and viridicatol. It also produces the mouldy smelling compounds geosmin and 2-methyl-isoborneol, and a series of specific orange to red pigments on yeast extract sucrose agar, hence the epithet discolor. P. discolor resembles P. echinulatum morphologically but on basis of the secondary metabolites is also related to P. expansum, P. solitum and P. crustosum. Introduction The mycotoxin chaetoglobosin C was first described from Chaetomium globosum (Sekita et al. 1973), but was later also found in fungi identified as Penicillium aurantiovirens, isolated from weevil-damaged pecans in Georgia (Springer et al. 1976). Frisvad and Filtenborg (1983) observed that these Penicillium strains have rough conidia and were similar to a strain isolated by them from an Israelian radish, which because of the morphological resemblance to P. hordei Stolk (rough conidia, synnemata formation) was first allocated to a provisional subgroup IV of P. hirsutum.this placement was made due to its good growth on creatine-sucrose agar, dark green conidia, orange red reverse colours on yeast extract sucrose agar and the specific profile of secondary metabolites (Frisvad & Filtenborg 1983). The strain also shows strong resemblance to P. echinulatum based on the dark rough conidia and it was later allocated to that species as chemotype II (Frisvad & Filtenborg 1989). A large number of isolates of this taxon have since been found especially on walnuts, hazelnuts, acorns, onions, and hard cheese and the fungus is described here as a new species. Materials and methods Isolates studied (Table 1) were three point inoculated on Czapek agar (Cz), Czapek yeast autolysate (CYA) agar, malt extract agar (MEA), 2% malt extract agar (MA), oat meal agar (OA, creatine-sucrose agar (CREA), yeast extract sucrose (YES) agar for one week at 25 C, but also on CYA for one week at 5 and 37 C (for media formulations, see Samson et al. 1995). Colony diameters were recorded after one week of growth, development of orange red pigments were examined from day 5 to day 14 after inoculation. All isolates were examined by TLC using the agar plug method (Filtenborg et al. 1983) in two different eluents. The secondary metabolites were visualized by anisaldehyde spray and 48% sulphuric acid spray (Frisvad & Filtenborg 1983; Cole & Cox 1981). Authentic standards of chaetoglobosin A (Jim Gloer, University of Iowa, USA), chaetoglobosin C (Sigma Chemical

2 120 Table 1. Isolates examined CBS = IMI = LHR9 = IBT 5738 = IBT (ex type): ex Radish, Israel CBS = IBT ex cheese, the Netherlands CBS = IBT 3904 = IBT 4231 = IBT 5734 = IBT 5920 ex Corylus avellana, Denmark CBS = DS 18342, isolated from cheese, Italy CBS = DS 19932, isolated from cheese, The Netherlands CBS = DS 19962, isolated from air in cheese factory, Belgium CBS = DS 19965, isolated from cheese, Austria CBS = DS 20033, isolated from cheese, France ATCC = IBT 4232 = IBT 5733 ex weevil damaged pecans, Georgia, USA ATCC = IBT 3088 = IBT 4229 = IBT 5730 ex weevil damaged pecans, Georgia, USA IBT 3086 = 4224 = 5744 ex Helianthus tuberosus, Denmark IBT 3089 = 4223 = 5731 ex onion, Denmark IBT 3090 = SA1 ex cheese The Netherlands IBT 3179 = IBT 3769 = IBT 5736 = IBT 4237 ex onion, Denmark IBT 3185 = 4228 = 5740 ex pecan nut IBT 3187 = IBT 4220 = IBT 5735 ex walnut, Denmark IBT 3557 = 4227 = 5737 ex apple, Lyngby, Denmark IBT 4512 = 4522 = 245,12 BMI IBT 5740 walnut, India IBT 11513, and ex acorn, Lyngby, Denmark IBT ex walnut, France IBT and ex cheese, Denmark IBT ex citrus fruit, Korea IBT = TM IBT ex Lechuguilla cave, Carlsbad, New Mexico, USA IBT K5, flower bulb, Denmark IBT ex blue cheese, France IBT = RMF 9950, IBT = IBT ex black walnut, North Carolina, USA Co.), palitantin, cyclopenin, cyclopenol, viridicatin, 3- methoxyviridicatin, cyclopeptin, dehydrocyclopeptin (from D.C. Aldridge, ICI, Macclesfield, Great Britain & A.G. Kozlowski, Puschino, Russia) were used to confirm identity. All isolates were also analyzed by reversed phase HPLC with diode array detection according to Frisvad and Thrane (1987, 1993) with extracts from two plates of each of CYA, MEA, YES, YES made from Sigma Y yeast extract, and OA. Identity of the compounds was only accepted if the compounds detected had the same UV spectra and retention indices as authentic standards. Description Penicillium discolor Frisvad and Samson, sp.nov. Figures 1 2. Coloniae in agaro YES dicto post 7 dies mm diam., pigmento laete aurantio vel rubro diffundente. In agaro creatino et sucrosio confecto bene crescit, nec 37 C crescere potest in ullo medio. In agaro CYA dicto 5 C 3 6 mm diam. post 7 dies. Conidia globosa vel subglobosa, (3 )3.5 4 m diam., obscure glauco-grisea vel viridi-grisea, aggregata raro ad colorem atrovenetum vertentia; conidiophora asperata, terverticillata, stipites m. Unus vel raro duo rami asymmetrice ad stipitem appressi; metulae cylindricae, asperatae, m; phialides lageniformes, m, collulo brevi praeditae. Chaetoglobosinum A et C, palitantinum, cyclopeninum, cyclopenolum, viridicatolum et alia viridicatina, geosminum, 2-methyl-isoborneolum producuntur. Types Herb. IMI , cultures ex type IMI = CBS = FRR 2933 = IBT 5738, isolato e Raphano sativo, Israel. Diagnosis. The species can be distinguished from other Penicillia by the combination of the following

Figure 1. Nomarski phase contrast micrographs of three isolates of Penicillium discolor. a c. CBS 474.

3 Figure 1. Nomarski phase contrast micrographs of three isolates of Penicillium discolor. a c. CBS (type isolate); d f CBS ; g i. CBS Magnifications: a, d and g. 600, all others

122 Figure 2. Scanning electron micrographs of critically point dried specimens a c. CBS 474.84, a. 1900, b., 4000, c. 3500 ; d. CBS 221.92, 3300. characters: globose to subglobose (3.

4 122 Figure 2. Scanning electron micrographs of critically point dried specimens a c. CBS , a. 1900, b., 4000, c ; d. CBS , characters: globose to subglobose (3.5 4 m) dark green rough walled conidia, terverticillate asymmetric penicilli with rough-walled stipes, synnemata production on malt agar (though not always pronounced), bright orange to red pigments diffusing on YES agar, production of chaetoglobosin A and C and other chaetoglobosins, palitantin, cyclopenin, cyclopenol, viridicatol and other viridicatins, geosmin, 2-methylisoborneol, and good growth on creatine sucrose agar. Several unknown metabolites were detected and indi-

5 123 Table 2. Production of secondary metabolites by isolates of P. discolor, as identified by HPLC-DAD CBS (T): Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met A, B, OY, XYX IBT 15185: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met A, B, cf. austdiol, IBT 15186: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met A, B, OY IBT 13945: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met A, B, OY, a naphthopyrone IBT 3187: Cha A, B, C, cyp, dec, col, vol, vin, pal, or-met IBT TM93045: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met A, CBS : Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met A, a naphthopyrone IBT 14472: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met A2, XYX, CO IBT 11513: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, a naphthopyrone, met OY IBT 11626: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, a naphthopyrone, met OY IBT acorn 13: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, a naphthopyrone, met A2, OY IBT 11724: Cha A, B, C, pal, or-met, met OY, XYX IBT 11725: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met OY, XYX IBT 3089: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met XYX IBT 3090: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met XYX ATCC 32000: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met ATCC 32002: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met IBT 11459: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met OY IBT : Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met A2, OY, IBT 4512: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met OY, a naphthopyrone IBT 6289: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met OY, a naphthopyrone IBT 6219: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met A2 IBT 6288: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, a naphthopyrone, met A2, OY IBT 6286: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, a naphthopyrone, met A2, OY, IBT 6287: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met A2, OY IBT 3086: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, met A, B, cf. austdiol IBT 16218: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met, a naphthopyrone IBT 16126: Cha A, B, C, cyp, dec, col, cin, vol, vin, pal, or-met Cha = Chaetoglobosin; cyp = cyclopeptin; dec = dehydrocyclopeptin; col = cyclopenol, cin = cyclopenin; vol = viridicatol; vin = viridicatin; pal = palitantin, or-met = orange metabolites, met = metabolite. cated in Table 2 as cf. austidiol, a naphthopyrone and metabolites A, A2, B, OY and XYZ. Description. Conidia globose to subglobose, (3 ) m, dark glaucous-gray to artemisia green, or occasionally approaching nickel or pea green en masse, the conidiophores are rough-walled, terverticillate, stipes m m. One ramus or rarely two rami are placed asymmetrically and appressed to the main stipe. Metulae cylindrical, rough-walled, m m. Phialides flask-shaped with short necks, 8 10 m m. Colonies on Czapek agar (7 days, 25 C) are mm diam., velutinous with floccose mycelium in the centre, plane or sulcate. Good sporulation, mycelium white and conidia dark glaucous-gray or artemisia green en masse. Reverse cream coloured to light brown, exudate clear to orange, soluble pigment absent. Colonies on CYA (7 days, 25 C) are mm diam., mostly velutinous, floccose or fasciculate, sometimes sulcate. Good sporulation, mycelium white and conidia artemisia green to nickel, dusk or pea green en masse. Reverse light brown with a dark centre, exudate orange or absent, soluble pigment absent. Clear to yellow exudate produced. Colonies or MEA or 2% MEA (7 days, 25 C) are mm diam., velutinous to strongly fasciculate, flat not sulcate. Good sporulation, mycelium white and conidia celandine green or sage green en masse. Reverse light green with a yellowish centre, exudate and soluble pigment absent. Colonies on YES (7 days, 25 C) are mm diam., velutinous to floccose, sulcate, good sporulation and a bright yellow reverse. In 6 to 12 days a bright orange to red diffusible pigment is produced. Very good growth on creatine-sucrose agar with acid and subsequent base production. No growth on

6 124 Table 3. Differences and similarities between P. discolor and related species (from Frisvad & Filtenborg 1989; Svendsen & Frisvad 1994; Larsen & Frisvad 1995a; Lund 1995) P. discolor P. echinulatum P. solitum P. palitans Growth on creatine Dark green conidia Rough conidia +/ /(+) - Palitantin /- Viridicatins /- Chaetoglobosins Territrems Penechins Compactins Cyclopiazonic acid Fumigaclavines Geosmin methyl-isoborneol Isopentanol Orange metabolite any media at 37 C. Colonies 3 6 mm diam. on CYA at 5 C after one week. Results and discussion Morphological differentiation All isolates of P. discolor produce rough conidia, however occasionally the roughending of the conidia was delayed compared to P. echinulatum and/or less pronounced (Figures 1, 2). The conidiophore stipes are not always distinctly roughened on malt agars. Colonies are slightly to strongly fasciculate on malt extract agars. P. discolor shows a resemblance to several creatine positive Penicillia, especially P. solitum, P. echinulatum, P. palitans, P. expansum, andp. crustosum. Someof the differences between the creatine positive species with dark green conidia are listed in Table 3. Chemical differentiation Isolates of P. discolor have been compared with all other terverticillate Penicillia using either profiles of secondary metabolites (Svendsen & Frisvad 1994) or profiles of volatiles (Larsen & Frisvad 1995a, b) and found to be distinct in both studies.p. discolor and P. crustosum are the only terverticillate Penicillium species that are able to produce geosmin and 2-methyl-isoborneol both volatiles known to have the musty/mouldy odours. They also both produce isobutanol and isopentanol, although P. crustosum also produces dimethyldisulphide, styrene and several esters (Larsen & Frisvad 1995a). When the volatile profile of P. discolor was compared to the other terverticillate Penicillia, it shared most volatiles with P. roqueforti var. carneum (Larsen & Frisvad 1995b). However, if it was compared using non-volatile secondary metabolites (Svendsen & Frisvad 1994), it showed greatest similarity to P. solitum and P. echinulatum. All isolates of P. discolor examined produced chaetoglobosin A and C (Table 2). This was confirmed by comparison to authentic standards in different TLC eluents using anisaldehyde as spray reagent (Frisvad & Filtenborg 1983) and by HPLC diode array detection (Frisvad & Thrane 1987). The production of chaetoglobosin A and C is only otherwise seen from P. expansum (Frisvad & Filtenborg 1989), another frequently occurring fungus on nuts. Chaetoglobosin C, however, appears to be one of the minor members of a whole series of chaetoglobosins in P. discolor. A comparison with an extract of Chaetomium globosum indicated that another major product in P. discolor was chaetoglobosin B, but no standard was available for this compound. The production of palitantin, also confirmed by comparison with an authentic standard, was shared with P. solitum, P. palitans, P. commune and P. echinulatum. This secondary metabolite was originally

7 125 isolated from P. palitans but was also isolated from a strain identified as P. frequentans (Turner 1971). The production of viridicatins is a feature of all strains of P. discolor examined, but these metabolites are quite common in the terverticillate Penicillia such as P. crustosum, P. solitum, P. palitans, P. echinulatum, P. vulpinum, P. freii, P. polonicum, P. aurantiovirens, P. cyclopium, P. neoechinulatum and P. scabrosum (Frisvad & Filtenborg 1989; Frisvad et al. 1990; Lund & Frisvad 1994). The most specific feature of P. discolor is the production of a number of yellow, orange-red metabolites with UV spectra not seen in any other Penicillium species. These metabolites are produced on media containing high concentrations of sugar, i.e. YES agar. Ecology P. discolor is particularly common on nuts, both from commercial and non-commercial sources. It has been found on hazelnuts, walnuts, black walnuts, pecans, and acorns (Table 1). It has also been found frequently in rhizosphere soil of onions, Jerusalem artichokes, radishes, Iris bulbs, and other plants parts found under soil. It also occurs on hard cheese. Thus its preferred habitats are rich in protein and often also lipid rich. It has not yet been found on cereals or any other sources and until now it has been found in temperate and subtropical climates in Europe, Israel and USA. The isolates found to produce chaetoglobosin C by Springer et al. (1976) were from weevil-damaged pecans, so a connection to certain insects cannot be excluded. All strains of P. discolor produced cyclopenin and cyclopenol. A strain of P. solitum, NRRL 6233, also a good producer of cyclopenin and cyclopenol, was isolated from a pecan that had been injured by a Southern green stink bug (Nezara viridula L.)(Cutleret al. 1984). Cyclopenin and cyclopenol had a strong effect on wheat coleoptiles, but also on other plants and insects (Cutler et al. 1984; Paterson et al. 1990). The production by P. discolor of both geosmin, 2- methyl-isoborneol and several other terpenes, alcohols and esters plus several insect toxic metabolites may suggest different interactions with animals. Toxicology Ethyl acetate extracts of the isolates of P. discolor from weevil-damaged pecans (ATCC ), grown on wheat, were shown by Springer et al. (1976) to be toxic to rats when introduced orally. The major toxic principle found was chaetoglobosin C, but toxicity tests with the pure compound were unsuccessful as the toxin was insoluble in water (Cole & Cox 1981). Chaetoglobosin A and the other chaetoglobosins have been shown to be cytotoxic (Sekita et al. 1973, 1976, 1977; Umeda et al. 1975) and acutely toxic to rats and mice (Ohtsubo et al. 1978). The consistent presence in extracts of both chaetoglobosin A and C and other chaetoglobosins, palitantin, cyclopenin, cyclopenol, viridicatin and viridicatol, all soluble in ethylacetate, indicates that the mixture of some of those compounds is toxic. No data are available on the production of these secondary metabolites on either nuts, vegetables nor cheese. None of the metabolites found are considered important mycotoxins (Cole & Cox 1981; Smith & Moss 1985). The best substrate for chaetoglobosin production was CYA and YES agar. On those media chaetoglobosin A is the major metabolite in that biosynthetic family. Acknowledgements The authors thank Prof. Walter Gams for providing the Latin diagnosis. References Cole RJ & Cox RH (1981) Handbook of toxic fungal metabolites. Academic Press, New York Cutler HG, Crumley FG, Cox RH, Wells JM & Cole RJ (1984) The biological properties of cyclopenin and cyclopenol. Plant Cell Physiol. 25: Filtenborg O, Frisvad JC & Svendsen JA (1983) Simple screening method for molds producing intracellular mycotoxins in pure cultures. Appl. Environ. Microbiol. 45: Frisvad JC & Filtenborg O (1983) Classification of terverticillate penicillia based on profiles of mycotoxins and other secondary metabolites. Appl. Environ. Microbiol. 46: Frisvad JC & Filtenborg O (1989) Terverticillate penicillia: chemotaxonomy and mycotoxin production. Mycologia 81: Frisvad JC & Thrane U (1987) Standardized high-performance liquid chromatography of 182 mycotoxins and other fungal metabolites based on alkylphenone indices and UV-VIS spectra (diode array detection). J Chromatogr. 404: (1993) Liquid chromatography of mycotoxins. In: Betina V (Ed.) Chromatography of mycotoxins: techniques and applications. Journal of Chromatography Library 54. Elsevier, Amsterdam, pp Frisvad JC, Samson RA & Stolk AC (1990) A new species of Penicillium, P. scabrosum. Persoonia 14: Larsen TO & Frisvad JC (1995a) Characterization of volatile metabolites from 47 Penicillium taxa. Mycol. Res. 99:

8 126 (1995b) Chemosystematics of Penicillium based on profiles of volatile metabolites. Mycol. Res. 99: Lund F (1995) Diagnostic characterization of Penicillium palitans, P. commune and P. solitum. Letters in Applied Microbiology 21: Lund F & Frisvad JC (1994) Chemotaxonomy of Penicillium aurantiogriseum and related species. Mycol. Res. 98: Paterson RRM, Simmonds MSJ & Blaney WM (1990) Mycopesticidal effects of characterized extracts of Penicillium isolates and purified secondary metabolites (including mycotoxins) on Drosophila melanogaster and Spodoptora littoralis. J. Invert. Pathol. 50: Ohtsubo K, Saito M, Sekita S, Yoshihira K & Natori S (1978) Acute toxic effects of chaetoglobosin A, a new cytochalasan compound produced by Chaetomium globosum, on mice and rats. Jpn. J. Exp. Med. 48: Samson RA, Hoekstra ES, Frisvad JC & Filtenborg O (Eds) (1995) Introduction of food-borne fungi. Centraalbureau voor Schimmelcultures, Baarn Sekita S, Yoshihira K, Natori S & Kuwano H (1973) Structures of chaetoglobosins A and B, cytotoxic metabolites of Chaetomium globosum. Tetrahedron Lett. 14: Sekita S, Yoshihira K, Natori S & Kuwano H (1976) Structures of chaetoglobosins C, D, E, and F, cytotoxic indol-3- yl-[13]cytochalasans from Chaetomium globosum. Tetrahedron Lett. 17: (1977) Chaetoglobosins G and J, cytotoxic indol-3-yl- [13]cytochalasans from Chaetomium globosum. Tetrahedron Lett. 18: Smith JE & Moss MO (1985) Mycotoxins. Formation, analysis and significance. John Wiley & Sons, Chichester Springer JP, Clardy J, Wells JM, Cole RJ, Kirksey JW, MacFarlane RD & Torgerson DJ (1976) Isolation and structure determination of the mycotoxin chaetoglobosin C, a new [13] cytochalasan. Tetrahedron Lett. 17: Svendsen A & Frisvad JC (1994) A chemotaxonomic study of the terverticillate Penicillia based on high performance liquid chromatography of secondary metabolites. Mycol. Res. 98: Turner WB (1971) Fungal metabolites. Academic Press, London Umeda M, Ohtsubo K, Saito M, Sekita S, Yoshihira K, Natori S, Udagawa S, Sakabe F & Kurata H (1975) Cytotoxicity of new cytochalasans from Chaetomium globosum. Experienta 31:

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